Image based cell screening is used in a variety of applications including pharmaceutical drug discovery, genomics and basic research and is known by several names such as high content screening, phenotypic screening or chemical genetics. The key requirements in cell screening are that the entire screening process of image acquisition, image analysis and data management needs to be fully automated, which requires robust imaging and image analysis techniques, and that the process is as non-invasive as possible to ensure that any biological changes observed in cells are not due to any adverse effects on cells, in whole or in part, by the screening process being used. Many previous image based cell screening methods have been invasive to varying extents.
Cell screening to date has generally been done using fluorescence microscopy where specific molecules or proteins of interest in cells are tagged with fluorescent probes or proteins and the temporal or spatial location of fluorescently labelled reporter molecules imaged and analyzed. However, fluorescence based imaging methods are invasive and may give incomplete information about cells.
There are inherent limitations to using fluorescent reporters and fluorescence illumination in cell screening with respect to maintaining the integrity of the cell and the reporter, particularly when living cells are imaged. Fluorescent methods may be toxic to cells where the toxicity may result from the fluorescent tag itself being toxic to cells; from damage to the cell caused by absorption of the excitation light by the fluorescent tag; or by damage to the cell caused by absorption of excitation light by natural components of the cells. Toxicity damage may also result from the generation of oxygen radicals or other like substances. The consequence is that the long-term survival of cells may be compromised by the necessity of using fluorescent approaches.
An additional limitation of fluorescence imaging results from photobleaching. Photobleaching is used to refer to the fluorescent excitation and resultant photochemical destruction of a fluorescent molecule when under fluorescent illumination. When photobleaching of a fluorescent reporter molecule in cell screening occurs, this may complicate the observation of the fluorescent molecules, since they will eventually be destroyed by the light exposure necessary to stimulate them into fluorescing. This may be especially problematic in time-lapsed microscopy.
Previous cell screening methods also generally employ dyes that bind to deoxyribonucleic acid (DNA). The DNA dye is excited by a different wavelength of light from that used to excite the fluorescent reporter molecules of interest. Commonly used dyes such as DAPI and Hoechst are excited at ultraviolet (UV) wavelengths that damage DNA and are injurious to cells. The main purpose of the DNA dye is to help in identifying the location of the nucleus, nuclear membrane or chromosomes. The location of the nucleus, an important landmark within the cell, is then used by image processing algorithms to provide positional information about other compartments of the cell and the location of fluorescent reporter molecules in the cell. The limitation of this technique is that these DNA-binding dyes, and the UV light used to excite them, are highly toxic to living cells and therefore highly invasive.
It is, therefore, desirable to provide a novel darkfield imaging system and method for automated screening of cells.